PROJECT ABSTRACT The repair of DNA double strand breaks (DSB) is critical for cell survival and the maintenance of genome stability. Most DSBs in human cells are repaired the classic non-homologous end joining (NHEJ) pathway, although a homology-dependent pathway that operates predominantly in the S and G2 phases of the cell cycle also plays an important role in DSB repair. Inactivation of either one of these pathways results in with increased cancer incidence because of increased genome instability. Furthermore, they appear to be frequently inactivated or dysregulated in cancer cells with defects in the homology-dependent pathway conferring sensitivity to PARP inhibitors. In this competitive renewal application, we are continuing to focus on the DNA ligase IV (LigIV)- dependent classic NHEJ pathway. Although studies by the Tomkinson laboratory and many others have provided detailed mechanistic insights into the mammalian and yeast NHEJ pathways, the mechanisms by which both strands of DSB ends are joined and the core NHEJ factors are released from ligated DNA not been definitively elucidated. In Aim 1, employing a novel assay that enables us to simultaneously monitor ligation of both strands and the association of NHEJ factors with the DNA, we will test the hypotheses that the ligations of both strands is co- ordinated by two molecules of LigIV and the ring-shaped Ku heterodimer remains topologically linked to the DNA duplex following ligation. The activity of the classic NHEJ pathway is a major determinant of radiosensitivity. In the past funding period, we showed that some cancers, identified by elevated levels of PARP and DNA ligase III, have reduced classic NHEJ activity. There is also emerging evidence that the expression levels of LigIV correlate with radioresistance and that radioresistant cancers with activated Wnt signalling have elevated levels of LigIV. This prompted us to identify three selective LigIV inhibitors by screening a library composed predominantly of FDA approved drugs. In Aim 2, we will further characterize these inhibitors and determine structure-activity relationships. Active compounds will also be evaluated for LigIV- dependent activity in cell-based assays. In Aim 3, compounds with LigIV-dependent activity in cell-based assays will be further characterized and used as probes to determine the effect of inhibiting classic NHEJ in non-malignant and cancer cells. In addition, the ability of the LigIV inhibitors to enhance the efficacy of ionizing radiation in reducing tumor growth will be evaluated in mouse xenograft studies with radiosensitive and radioresistant cancer cell lines. We envision that the proposed studies will provide novel insights into the mechanism and contribution of LigIV- dependent NHEJ to DSB repair in non-malignant and cancer cells and will generate novel reagents to evaluate the utility of LigIV inhbitors in enhancing the efficacy and fidelity of gene editing and as radiosensitizers that increase the effectiveness of tumor-targeted radiation therapy.